Project Summary/Abstract Muscle dysfunction from disease, trauma or aging, a major health care burden not adequately alleviated by available therapies, could be greatly reduced by treatments that directly address regeneration pathophysiology by capitalizing on muscle stem cells. Satellite cells comprise a population of progenitor cells that contains muscle stem cells resident within skeletal muscle, and are the primary cellular source for muscle injury repair and homeostatic turnover. Satellite cells are therefore likely focal points of vulnerability in muscle aging and disease states, and in turn are promising therapeutic targets. Methods for isolating and transplanting mouse satellite cells (MuSC) have been well developed, leading to fundamental insights. However, the field of human satellite cell biology has progressed more slowly. The heterogeneous nature of satellite cells in health and disease is poorly understood, and which subpopulations of satellite cells are capable of functions of self- renewal or differentiation is unknown. Recent efforts to extend MuSC biology to human have yielded new approaches to characterize endogenous human satellite cells (HuSC), paving the way for experimental investigations and eventual clinical applications. Deeper knowledge of the heterogeneous nature of human satellite cells, and specifically of the muscle stem cells within the satellite cell pool, is needed to understand how the reserve of stem cells in human muscles changes in aging and disease states, and to develop targeted therapies for muscle disorders. Our long-term objective is to develop regenerative clinical applications using and targeting HuSCs. To better understand the target cell type, this proposal will test the hypothesis that human satellite cells express heterogeneous transcriptional signatures and functions, and that heterogeneity changes during regeneration and aging. Our experiments will use healthy skeletal muscle from adult men and women and are based on preliminary data showing that postnatal human muscle progenitors exist as a distinct pool of satellite cells, which in turn have heterogeneous phenotypes. In Aim 1, we seek to distinguish heterogeneous HuSC populations, and discern relationships of transcriptional identities to regeneration and differentiation properties. Single-cell transcriptome data will be obtained from HuSCs to characterize heterogeneity and heterogeneous subpopulations will be examined in transplantation and regeneration assays in vivo and differentiation assays in vitro. In Aim 2, we will determine how cellular and organismal aging, and activity of aging-related pathways affect HuSC heterogeneity and function. Intrinsic age-related changes in HuSC function will be determined by comparing single-cell transcriptome data and regeneration of HuSCs from young, middle-aged and elderly adults, and by interfering with canonical aging pathways. This innovative combination of in vivo regeneration studies of HuSC with single-cell transcriptomic analysis to investigate endogenous muscle progenitor subpopulations will advance our understanding of endogenous human muscle stem cells, which is essential to develop clinical applications that aim to treat skeletal muscle disorders.